Measurement of procoagulant platelets in platelet-rich plasma by flow cytometer for the diagnosis of heparin-induced thrombocytopenia

Introduction Heparin-induced thrombocytopenia (HIT) is caused by anti- PF4/Heparin IgG antibodies, which activates platelets and leads to thrombocytopenia and thrombosis. The diagnosis of HIT can only be confirmed by using functional assays such as the Heparin-Induced Platelet Activation assay (HIPA assay). However, functional assays are technically demanding and routinely available only in specialized laboratories. The aim of the current study was to establish a flow cytometer-based method to detect procoagulant platelets using platelet-rich plasma (PRP) for the diagnosis of HIT.

Method Sera samples from patients with HIT were incubated with PRP from healthy donors for different durations (30, 60, 90 minutes). Procoagulant platelets were determined by double expression of P-selectin (CD62P) and phosphatidylserine (PS) externalization by flow cytometry. CD32a-mediated cross-linking and platelet stimulation with TRAP-6 and Convulxin were used as positive controls.

Results Sera from HIT-diagnosed patients but not from the control-group induced a significant increase in the procoagulant platelet subpopulation in the presence of 0.2 U/mL heparin, compared to 100 U/mL treated cells (% double positive CD62P/Annexin, 100 U/mL vs. 0.2 U/mL Mean±SEM: 1.2±1.1 vs 18.5±8.1, p=0.0021). The optimal incubation time was detected after 60 minutes. A donor dependency of the flow cytometric method was not observed (% double positive CD62P/Annexin, control vs. HIPA+, Mean±SEM: 0.7±0.59 vs. 19.0±3.2, p=0.0129). In addition, the use of washed platelets and PRP with HIT-sera showed comparable results in the flow cytometric analysis (% double positive CD62P/Annexin, PRP vs. washed platelets Mean±SEM: 34.2±6.3 vs 30.1±2.2, ns).

Conclusion Our data suggest that flow cytometry-based protocol using PRP can be suitable to detect the ability of HIT antibodies to induce procoagulant platelets by flow cytometry. An ongoing study is currently investigating the clinical implementation of this protocol in the diagnostic of HIT.

Conflict of Interest

I have no potential conflict of interest to report.

Publication History

Article published online:
20 February 2023